An Open-Source Mouse Chronic EEG Array System with High-Density MXene-Based Skull Surface Electrodes

Electroencephalography (EEG) is an indispensable tool in epilepsy, sleep, and behavioral research. In rodents, EEG recordings are typically performed with metal electrodes that traverse the skull into the epidural space. In addition to requiring major surgery, intracranial EEG is difficult to perform for more than a few electrodes, is time-intensive, and confounds experiments studying traumatic brain injury. Here, we describe an open-source cost-effective refinement of this technique for chronic mouse EEG recording. Our alternative two-channel (EEG2) and sixteen-channel high-density EEG (HdEEG) arrays use electrodes made of the novel, flexible 2D nanomaterial titanium carbide (Ti3C2Tx) MXene. The MXene electrodes are placed on the surface of the intact skull and establish an electrical connection without conductive gel or paste. Fabrication and implantation times of MXene EEG electrodes are significantly shorter than the standard approach, and recorded resting baseline and epileptiform EEG waveforms are similar to those obtained with traditional epidural electrodes. Applying HdEEG to a mild traumatic brain injury (mTBI) model in mice of both sexes revealed that mTBI significantly increased spike–wave discharge (SWD) preictal network connectivity with frequencies of interest in the β-spectral band (12–30 Hz). These findings indicate that the fabrication of MXene electrode arrays is a cost-effective, efficient technology for multichannel EEG recording in mice that obviates the need for skull-penetrating surgery. Moreover, increased preictal β-frequency network connectivity may contribute to the development of early post-mTBI SWDs.


Significance Statement
Electroencephalography (EEG) is a critical technique used to study neurological activity in rodents.Commonly used EEG procedures require time-consuming skull-penetrating surgeries that may confound the experiments.Here we provide a cost-effective solution for obtaining two-channel (EEG2) and high-density EEG (HdEEG) recordings on the skull surface thus avoiding major surgery.We compared this HdEEG system to traditional EEG recordings and then used it to determine the effects of mild traumatic brain injury on preictal network connectivity.This novel open-source EEG system will contribute to the electrophysiological characterization of mouse behaviors and seizure activity.
Human EEG electrodes are metal cups filled with an electrolyte gel that establishes electrical contact with the scalp and must be repeatedly replaced during extended recordings due to gel drying (Searle and Kirkup, 2000).In addition to electrolyte drying, gel-filled cup electrodes cannot be used for chronic rodent EEG recordings due to the difficulty in attaching them to the scalp/skull and the propensity of the electrolyte to bridge adjacent electrodes on their small skulls.Therefore, current rodent EEG studies typically use 0.1-1.0mm diameter metal electrodes inserted via cranial burr holes into the epidural space to establish electrode/electrolyte contact and to secure them for chronic recording.Drilling cranial burr holes is a major surgery that could potentially alter physiology.Skull-penetrating surgeries are difficult in young rodents with thin skulls, and they confound experiments studying mild traumatic brain injuries (mTBI), which are not associated with skull fractures.Epidural HdEEG experiments done with homemade arrays involve laborious fabrication and implantation procedures (Wasilczuk et al., 2016;Ding et al., 2019).While HdEEG experiments can also be performed using commercially available ultrathin 16-32 channel electrode arrays that lie on the skull surface (Choi et al., 2010;Jonak et al., 2018Jonak et al., , 2020)), they still require the placement of three epidural anchoring screws and their cost is prohibitive.There is a need for inexpensive, easily fabricated EEG2 and HdEEG arrays that can be implanted without skull injury.
In this open-source EEG2/HdEEG method, investigators design thin flexible printed circuit boards (PCBs) with electrode pads overlying cortical regions of interest.The electrode pads are then functionalized with soft contacts made with titanium carbide (Ti 3 C 2 T x ) MXene, a biocompatible (Dai et al., 2017), flexible 2D nanomaterial that provides electrical contact between the electrode pad and the skull without the need for electrolyte gel or paste (Driscoll et al., 2021).The EEG2/ HdEEG arrays are chronically affixed to the skull surface with UV-curable cement without the need for any skull burr holes.We validated this system by measuring normal and epileptiform EEG2 signals obtained with traditional metal epidural electrodes and MXene electrodes and comparing the spatiospectral distribution of preictal and epileptiform spectral density obtained with MXene HdEEG electrodes with those reported previously using epidural HdEEG arrays.Finally, we used MXene HdEEG arrays to determine the acute effects of mTBI on SWDs.Many mTBI patients have long-term neurological symptoms (Nelson et al., 2019), and elucidating the neurophysiological consequences of mTBI in rodents is a necessary step in developing effective mTBI therapies (Liang et al., 2022).

Materials and Methods
Animals.The protocols were approved by the (author university) Animal Care and Use Committee.We used adult mice (20 ± 3 weeks, 26.8 ± 3.8 g) in the DBA/2J background that heterozygously expressed a missense human epilepsy mutation in the GABA A receptor (Gabra1 +/A322D ) that are an established model of epileptiform SWDs (Arain et al., 2012(Arain et al., , 2015)).Mice had unlimited access to food and water and were housed in a temperature-and humidity-controlled environment with a 12 h lights-on/lights-off cycle with lights on at 0600 (Zeitgeber Time 0, ZT0).After all experiments, mice were killed by CO 2 asphyxia on the last day of recording and their skulls and brains were examined to evaluate for the presence of visible injury.
Electrode arrays.We compared MXene electrodes with traditionally used 1.0 and 0.1 mm diameter metal electrodes; 1.0 mm electrodes are more stable than 0.1 mm electrodes while the thinner 0.1 mm electrodes are more suitable for highdensity intracranial recordings.The 1.0 mm screw electrodes were commercial EEG2/EMG arrays from Pinnacle (catalog #8201), and the 0.1 mm electrodes were made from thin tungsten rods (Ding et al., 2019).
The PCBs for the 0.1 mm epidural tungsten EEG2 arrays were identical to the MXene arrays except that the electrode pads were 1.05 mm diameter rings with 0.75 mm holes.Five gold-plated electronic pin receptacle connectors (0.52 mm inner diameter, Mill-Max, 4428-0-43-15-04-14-10-0) were soldered to the electrode pads through the 0.75 mm holes and then strengthened with nonconductive epoxy.The 0.1 mm epidural electrodes were five 3 mm tungsten rods (254 µm diameter, A-M Systems 717200) cut to 3 mm and ground to a 100 µm tip with a Dremel rotary tool and then inserted through the pin receptacle connectors.
For both MXene and tungsten arrays, the wires connecting electrode pads were 150 µm wide.We designed the PCB to have pads for the attachment of either one four-position (EEG2) or two ten-position (HdEEG) rectangular connector sockets (Fig. 1A,B) to connect the arrays to the EEG amplifier.The use of the four-position EEG2 connector allows the array to interface with a commercial mouse EEG2 acquisition system (Pinnacle).Flexible PCBs using this design were manufactured on 0.2 mm polyimide by a commercial PCB prototyping company (PCBWay).
For the MXene arrays, a polyimide template with holes overlying the electrode pads was made using a CO 2 laser cutter.Ti 3 C 2 T x MXene aqueous dispersions were provided by Murata Manufacturing.A nonwoven, hydroentangled cellulose polyester (60-40%) blend substrate was infused with the Ti 3 C 2 T x MXene (20 mg/ml) and thoroughly dried.Circular electrode contacts were then cut out from the MXene-infused substrate using a biopsy punch to form circles (h 0.4 mm, d 500 µm).With the polyimide template placed over the PCB, the circular MXene contacts were applied to each electrode pad using conductive silver epoxy (CircuitWorks CW2400).Rectangular socket connectors were soldered to the PCB connector pads (four-position connector for EEG2 cut from six-position Samtec SFMC-103-T1-S-D; two ten-position connectors for HdEEG cut from twenty-position Samtec CLP-110-02-L-D-P-TR).
Samples of both the tungsten electrodes and MXene electrodes were characterized by electrochemical impedance spectroscopy using a Gamry Reference 600 potentiostat.Measurements were conducted at room temperature in 10 mM phosphate-buffered saline (PBS solution, pH 7.4) using a three-electrode configuration, with an Ag/AgCl reference electrode and a graphite rod counter electrode.Impedance spectra were collected at frequencies ranging from 1 Hz to 1 MHz with 10 mV rms AC voltage.
Surgery.Under isoflurane anesthesia, we made an incision in the dorsal scalp, cleaned and dried the skull, and visually centered the positioning hole over the bregma to ensure reproducible placement.For the epidural EEG2 arrays, the PCBs were attached to the skull with cyanoacrylate adhesive and dental cement.For the epidural recordings, a 29 Ga needle was inserted through the lumen of the electrode connectors to gently drill burr holes in the underlying skull.The 1.0 mm steel screw electrodes or 0.1 mm tungsten electrodes were inserted in the connectors with their tips in the epidural space.The MXene HdEEG arrays were placed on the skull, and a small amount of ultraviolet-curable adhesive (Visbella UF0008CR2P) was applied individually to each access hole while the nearby MXene electrode pads were held firmly against the skull to prevent the epoxy from seeping between the electrode and the skull.A UV light cured the epoxy.
Overpressure blast mTBI.We used a validated overpressure method for producing mTBI (Heldt et al., 2014;Reiner et al., 2014;Bu et al., 2016;Guley et al., 2016Guley et al., , 2019;;Jha et al., 2018;Honig et al., 2021;Mondal et al., 2021).The mice were anesthetized with 2-4% isoflurane in 100% O 2 throughout the procedure, first in an induction chamber and then via a nasal cannula while the mice were in the overpressure apparatus.After anesthesia induction, the mice were placed in a plastic holding tube, which was then inserted in a second plastic tube that is situated on an x-y table that can precisely position the skull 4 cm from the barrel of a paintball gun.The dorsum of the mouse skulls (at bregma) was positioned under 6 mm holes in the plastic tubes that align with the barrel and transmit the pressure blast to a precise location in the skull.The pressure of the paintball gun provided a single 40 psi pressure wave with a half-width duration of 7 ms; sham mice experienced the anesthesia and exposure to the apparatus without overpressure.After the sham/mTBI, we measured the righting time, a common immediate assessment of traumatic brain injury (TBI) severity (Kane et al., 2012;Gullotti et al., 2014).Immediately after the mTBI/sham, the mouse was placed on its side and the time until it stood on its paws was considered the righting time.
EEG recording and analysis.Continuous epidural (1.0 mm) and MXene EEG2/EMG recordings were performed in the (author university) Mouse Neurobehavioral Laboratory for 2 d starting 7 d after implantation using Pinnacle amplifiers (sampling 400 Hz).MXene HdEEG recordings and 0.1 mm epidural EEG2 studies were performed for 3 h using a Nicolet V32 amplifier sampled at 250 or 500 Hz 1, 2, and 3 weeks after surgery.Electrode impedances across the skull (MXene) were measured using Nicolet's proprietary protocol; because the outputs of the Pinnacle prefabricated 1.0 mm headmounts were bipolar, the epidural impedances of single 1.0 mm Pinnacle epidural electrodes could not be measured.
To record convulsive tonic-clonic seizures, mice were injected intraperitoneally with pentylenetetrazol (PTZ, Sigma-Aldrich), dissolved in saline (1 mg/ml, 7 mM).The mice were first injected with 40 mg/kg PTZ; if a convulsive seizure did not occur within 10 min, an additional 60 mg/kg was injected.
A reviewer blinded to mouse identity and treatment identified EEG segments for analysis.For the continuous EEG2/ EMG recordings, EEG/EMG spectrograms were generated using the EDFbrowser software.Awake epochs were identified as periods of high-power EMG and low-delta (≤4 Hz) EEG power, and epochs of slow wave sleep (SWS) were identified as periods of low-power EMG and high-delta EEG power (Louis et al., 2004).In addition, the logarithmic δ/ϴ spectral power ratios were calculated in 10 s windows in overlapping 1 s intervals using a fast Fourier transform with a Hanning taper and with δ frequencies of 1-4 Hz and ϴ frequencies of 5-7 Hz.Awake epochs had lower δ/ϴ spectral power ratios, and SWS epochs had higher δ/ϴ spectral power ratios.
To compare the EEG signal quality obtained from the two electrode types, we quantified the movement artifact during 30 min samples of wakefulness.A blinded reviewer experienced in identifying movement artifacts reviewed awake EEG obtained with 1.0 mm epidural (the left hemisphere referenced to the cerebellum) and MXene (M3 electrode −1.85 mm M/L, −0.25 mm A/P referenced to the midbrain) using the EDFbrowser software and marked the beginning and end of movement artifact.The seconds of movement artifact per hour of awake EEG were then calculated.
Five segments (10 s) of awake and SWS EEG without artifact or epileptiform discharges were chosen from each mouse for quantitative analysis.In addition, SWDs were identified as high-voltage rhythmic 6-8 Hz waveforms (Robinson and Gilmore, 1980;Sitnikova and Van Luijtelaar, 2007).Five spike-wave discharge (SWD) segments were chosen from each mouse from 5 s before to 2 s after SWD onset (t 0 defined as the time of the first SWD spike).
EEG analysis was performed using the FieldTrip MATLAB toolbox (Oostenveld et al., 2011).EEG2 and HdEEG were downsampled to a sample frequency (Fs) of 200 Hz and bandpass filtered (BP) between 0.5 and 55 Hz using a two-pass windowed-sinc finite impulse response filter (Widmann et al., 2015).For quantification of HdEEG preictal spectral power and network connectivity and for the comparison of MXene EEG signal on Days 7, 14, and 21, the EEG was also band-stop (BS) filtered at 60 Hz using a two-pass Butterworth notch filter.
As another method of comparing the SWD EEG2 spectral power between MXene and epidural 1.0 mm electrodes and to account for the voltage differences between these electrode types and days postimplantation, we Z-transformed the EEG2 traces before performing spectral analysis.We first used the MATLAB "detrend" function to remove the fitted mean from the data and then used the MATLAB "envelope" function to calculate the root mean square of the signal with a sliding window of 10 s.The Z-transformed signals were calculated as the detrended signals divided by the root-mean-square envelope.
EEG2 and HdEEG spectral power and HdEEG cross-spectral density were calculated using a two-cycle Morlet transform from 2 to 55 Hz.The EEG2 Morlet transforms were performed every 50 ms from 0.5 to 9.5 s of the awake background/ SWS and from 0.5 to 1.5 s of the ictal SWD and then averaged among those timepoints.The spectral power of PTZ-evoked convulsive seizures was calculated using the same Morlet transform every 50 ms of the first 5 s of the seizure.The HdEEG Morlet transforms were performed every 50 ms from 5 s before to 2 s after SWD t 0 .The spectral density and cross-spectral density measurements were averaged among the five awake/SWS/SWD segments from each mouse.
To compare SWD preictal increases in β-frequency spectral power recorded with MXene HdEEG with those obtained with previous studies, the preictal (0.10 to 1.00 s prior to t 0 ) β-spectral frequency was normalized to the preictal baseline (3.00-5.00s before t 0 ) as decibels (dB), which are calculated as follows: dB = 10 X log 10 P preictal P baseline where P preictal and P baseline are the preictal and baseline spectral power, respectively.We used the weighted phase lag index (WPLI), a network measure that is insensitive to volume conduction (Vinck et al., 2011), to determine the effects of mTBI on SWD preictal network connectivity.The HdEEG preictal Morlet transform crossspectral density measurements were averaged in overlapping 1 s bins spaced every 0.5 s from 1 to 5 s before SWD onset.The 2-30 Hz WPLI between each electrode pair was then calculated at these time points.A bootstrap method was used to calculate the WPLI 95% confidence interval and the WPLI values with confidence intervals that did not overlap zero were considered suprathreshold (McGuinness et al., 2022).The network node degree, the number of suprathreshold connections, was determined at each electrode.
Statistical analyses.Statistics were calculated using R for Windows version 4.1.3.The Shapiro-Wilk test was used to determine if variables were normally distributed.Fabrication and surgery times and differences in mTBI/sham righting times were compared using two-tailed t tests.Differences between epidural and MXene electrodes in movement artifact times were determined using the Wilcoxon signed-rank test and the median (Md), first and third quartiles (Q1, Q3), and bootstrapped confidence interval (CI) are presented.Perioperative deaths (within 7 d) were compared using the chi-squared test.Differences in the HdEEG preictal channel/frequency/time distribution of spectral density and network node degree were calculated using cluster-based permutation (CBP) analyses.CBP analysis is a nonparametric statistical method (Maris and Oostenveld, 2007) that addresses the multiple comparison problems inherent in analyzing numerous electrode, frequency, and time combinations by clustering nearby electrode, frequency, and times based on either initial repeated measure (comparing preictal and baseline spectral power) or independent (comparing mTBI with sham) t tests.The summed t score of the clusters was determined.The data sets were then randomly permuted 500 times, and the percentile of the initial summed t score versus the permuted t scores is the probability that the spatiospectral-temporal distributions differ.Here, the CBP analyses were run with spatial clusters including electrodes within 2 mm and a critical cluster statistic of 0.025; p values were appropriately adjusted to account for two-tailed comparisons.

Array fabrication and implantation
Top and bottom views of completed MXene HdEEG arrays are shown in Figure 1, C and D. Despite having 16, rather than 2 channels, the fabrication time of the MXene HdEEG arrays (97 ± 5 min) was significantly shorter than that of the epidural 0.1 mm EEG2 arrays (179 ± 57 min; p = 0.004).Impedance spectroscopy demonstrated that the epidural 0.1 mm and MXene electrodes had similar electrode/electrolyte impedances at frequencies between 1 and 1,000 Hz (Fig. 1E) with a maximal difference of only 3.1 kΩ at 1 Hz.Despite having a larger number of channels, the average duration of the implantation surgery for MXene HdEEG electrodes (55 ± 4 min, N = 7) was significantly shorter than that for epidural 0.1 mm EEG2 arrays (73 ± 4 min, N = 12, p < 0.001).The mice tolerated the implantation of all types of arrays.Two of the 31 MXene HdEEG implanted mice died in the perioperative period (within 7 d), and there were no perioperative deaths in the 0.1 mm (N = 14) or 1.0 mm (N = 6) epidural-implanted mice (χ 2 = 1.343, p = 0.51).

EEG2 recordings with epidural and MXene electrodes
We performed continuous 48 h EEG2 recordings using 1.0 mm epidural and MXene electrodes.EEG and EMG spectral density (Fig. 2A,B) were analyzed to identify periods of wakefulness (low δ power, low δ/ϴ power ratios, and high EMG power) and SWS sleep (high δ power, high δ/ϴ power ratios, and low EMG power).Differentiation between awake and SWS states was clear with both electrode types.SWS recorded with epidural electrodes had increased ϴ as well as δ power, and thus the SWS-associated increases in δ/ϴ power ratios were smaller for epidural electrodes than MXene electrodes.
Figure 2, C and F, presents the sample EEG2 traces obtained with epidural (C,D) and MXene (E,F) electrodes in wakefulness (C,E) and SWS (Fig. 2D,F).Raw spectrograms of awake and SWS EEGs are shown in Figure 2, G and H, with insets depicting the corresponding spectrograms of Z-transformed EEG.The raw spectrogram of awake EEG (Fig. 2G) shows that awake recordings by epidural and MXene EEG2 exhibit similar distributions of spectral density although the MXene electrodes (peak 4 Hz, 33,006 ± 5,201 µV 2 ) exhibit somewhat decreased power in frequencies above 6 Hz than the epidural electrodes (peak 6 Hz, 35,144 ± 7,619 µV 2 ).The larger contribution of lower frequencies during wakefulness in MXene electrodes is apparent in the spectrogram of Z-transformed EEG (Fig. 2G, inset).Compared with the awake state, the peak frequencies in SWS for both electrode types were lower (3 Hz) and of greater magnitude for both the epidural (86,382 ± 14,766 µV 2 ) and MXene (46,760 ± 5,253 µV 2 ), although there is a greater relative increase in epidural electrodes (146%) than in MXene electrodes (42%).
Next, we compared the EEG2 recordings of epileptiform SWDs obtained with 1.0 mm epidural and MXene electrodes during continuous EEG2 recording.SWDs obtained with both types of electrodes showed the typical spike, positive transient, and wave components reported previously in rodent absence seizures (Robinson and Gilmore, 1980;Sitnikova and Van Luijtelaar, 2007).Spectrograms of the SWD spectral density are shown in Figure 3C.The epidural 1.0 mm electrodes had a greater amplitude of raw spectral power than the MXene electrodes but both electrode types exhibited a broad peak of spectral density from 6 to 30 Hz reflecting the 6-8 Hz primary SWD frequency along with the first, second, and third harmonics.The inset in Figure 3C is the average SWD spectrogram with EEGs that were first Z-transformed and demonstrates that although SWDs have a higher voltage when recorded with epidural than MXene electrodes, they have the same distribution of spectral density.
No 1.0 mm epidural electrodes or MXene electrodes were lost during the 2 d continuous video-EEG monitoring recording.To compare the durability of MXene electrodes with 0.1 mm epidural electrodes, those that can be used for epidural HdEEG recordings, we made weekly 3 h EEG2 recordings of MXene (N = 11) and 0.1 mm epidural electrodes (N = 11) 1, 2, and 3 weeks after implantation.While 82% of the epidural 0.1 mm preparations lost at least one electrode within 3 weeks of surgery, only 18% of the MXene implants lost at least one electrode at 3 weeks.
Figure 4A depicts MXene electrode SWD recordings obtained from the same mouse 7, 14, and 21 d after electrode implantation.The SWDs in all three recordings are similar although the voltage of the one obtained at 21 d is somewhat lower than on Days 7 and 14.The mean spectral power tracings of SWDs obtained from mice at 7 d (N = 6), 14 d (N = 5) and 21 d (N = 6) are shown in Figure 4B.The distribution of SWD spectral power is similar at each timepoint although the spectral power at Day 21 is approximately 30% lower at all frequencies compared with the mice recorded on Days 7 and 14.The inset of Figure 4B depicts the mean spectral power of Z-transformed EEG recordings of SWDs and demonstrates similar spectral profiles at all three timepoints, a result that indicates that although the voltage of the 21 d recordings is less than that at other timepoints, the ratio of the SWD signal to background EEG variability remains unchanged.
MXene and 1.0 epidural electrodes were also durable during convulsive seizures; neither electrode type was lost during the convulsions.Tracings of PTZ-evoked tonic-clonic seizures were similar when obtained with epidural (Fig. 5A) and MXene (Fig. 5B) electrodes.The lower tracings in Figure 5, A and B, are the first 5 s of the convulsive seizures depicted on an expanded time scale to demonstrate that convulsive movements do not prevent the identification of EEG epileptiform waveforms.Figure 5C demonstrates the mean spectral power of the first 5 s of PTZ-evoked convulsive seizures recorded with epidural (N = 5 mice) and MXene (N = 5 mice) electrodes, and the inset is the spectral power of the Z-transformed EEG.The spectral profiles of PTZ-evoked convulsive seizure onset were similar when recorded 1.0 mm epidural and MXene electrodes; however, as expected, the absolute spectral power was lower for the MXene electrodes (peak 7 Hz, 1,29,917 ± 13,730 μV 2 ) than 1.0 mm epidural electrodes (peak 6 Hz 3,70,898 ± 87,240 μV 2 ).

MXene 16 channel HdEEG SWD recording
We next recorded 16-channel HdEEG using the MXene electrodes.Like the M3/M4 electrodes, the additional MXene HdEEG electrodes, with the exception of S5p and S6p, provided excellent EEG signals for at least 3 weeks (data not shown).However, the S5p and S6p electrodes in the lateral posterior region were more likely than the other HdEEG to become dislodged early after implantation (Week 1, with probabilities of 0.38 and 0.31, respectively).
Previous studies used epidural HdEEG electrodes to map the spatial distribution of SWDs (Ding et al., 2019), and thus we recorded SWDs with MXene HdEEG electrodes and compared the SWD spatial distribution with those of the prior reports.A HdEEG SWD tracing is depicted in Figure 6A, and a topology plot of the mean 6-8 Hz spectral power of the SWD onset (0.5-1.5 s after the first spike) is shown in Figure 6B.As seen previously with epidural HdEEG electrodes (Ding et al., 2019), the characteristic 6-8 Hz spikes and waves are recorded in all electrodes over the dorsal cortex, but with prominence in the frontal electrodes over the motor cortex.
Prior experiments with epidural electrodes also found small, but significant, preictal increases in β-frequency spectral density just prior to SWD onset (Sorokin et al., 2016;Ding et al., 2019), and thus we determined if these findings could be observed with MXene HdEEG electrodes.Figure 6C depicts the mean preictal (3.00-0.10s before t 0 ) β-frequency (13-30 Hz) normalized spectral density and demonstrates an increase in β-frequency spectral power starting at approximately 0.40 s prior to SWD onset, a finding consistent with previous results.Interestingly, there was also a brief decrease in spectral power in the frontocentral electrodes at approximately 1.00 s before the SWDs.CBP found a significant difference (p = 0.006) in β-spectral power between the baseline (3-5 s prior to SWD) and the preictal period (0.10-1.00 prior to SWD).Prominent frequency/time point CBP clusters included the decreased 13-30 Hz spectral power in the frontocentral electrodes at −1.00 s (Fig. 6D) and increased 13-22 Hz power in the posterior electrodes at −0.25 s (Fig. 6E).

Effect of mTBI on preictal network connectivity
A family history of epilepsy is a significant risk factor for the development of seizures after mTBI (Christensen et al., 2009).Possibly, mTBI exacerbates mild preexisting epileptic network.Since MXene HdEEG can record SWDs without intracranial burr holes that can potentially confound mTBI experiments, we used MXene HdEEG to determine the effects of mTBI on the SWD preictal network.One week after MXene HdEEG implantation, mice underwent a 2 h baseline recording followed by either mTBI or sham.The mTBI produced no difference in visible behavior or righting times (mTBI 107 ± 19 s, sham 113 ± 17 s).Subsequent HdEEG recordings at 1 d and 7 d after mTBI/sham demonstrated that mTBI did not disrupt MXene electrode recording quality.Postmortem examination of the brains found no visible evidence of injury in either group (data not shown).
To determine the effects of mTBI on SWD preictal network connectivity, we calculated the interelectrode WPLI and network node degree at frequencies 2-30 Hz from 1 to 5 s prior to SWD onset.While sham mice exhibited a decreased preictal network connectivity from the pre-sham recording to 1 d post-sham (Fig. 7A,C), mTBI increased preictal network connectivity (Fig. 7D).We used CBP to statistically compare sham and mTBI on changes in network node degree from 2 to 30 Hz and at times from 1 to 5 s prior to ictal onset (Fig. 7E, p = 0.02).Clusters in CBP testing were at 1.0 and 1.5 s prior to SWD onset and included β-frequencies (13-20 Hz) in almost all channels.Although there were also increases in network node degree on Day 7, these changes were not statistically significant (p = 0.06, data not shown).

Discussion
This study demonstrated high-quality skull surface EEG2 and HdEEG recordings using open-source MXene-based EEG arrays.The signals recorded with MXene electrodes were comparable to those obtained with epidural electrodes, albeit with an expected lower voltage and preferential attenuation of faster frequencies.MXene HdEEG recordings revealed that mTBI produced acute changes in preictal SWD connectivity, despite the absence of visible behavioral differences.These results demonstrate that the MXene skull surface electrode array is a novel, inexpensive tool that allows a minimally invasive mechanism to study mouse neurophysiology.

Comparison of MXene electrodes with current techniques
Compared with current epidural EEG2 and HdEEG methods (Wasilczuk et al., 2016;Ding et al., 2019), this open-source method of MXene electrode skull surface EEG eliminates the need for drilling skull burr holes.This advance represents a major refinement in animal care.Eliminating skull burr holes also mitigates potential confounding factors in rodent physiology experiments, especially those designed to study the effects of TBI or those investigating the physiology of young mice.MXene EEG array fabrication is more efficient than the epidural open-source techniques given that its layout and wiring are achieved using PCBs and its electrode pads are easily coated with MXene.Importantly, surgical implantation of MXene arrays is easier and more efficient than epidural electrodes since burr holes are not required and may thus be performed by laboratory personnel with less experience than those who typically perform intracranial surgeries.
Nanofabricated 16-and 32-channel skull surface HdEEG arrays are commercially available (NeuroNexus) and are substantially thinner (15 µm) than the ones produced by our open-source method.However, although the electrodes of the commercial arrays lie on the skull surface, chronic placement of this array still requires three skull-penetrating anchoring screws (Jonak et al., 2018) and thus is problematic for mTBI studies and experiments using young mice with thin skulls.Another substantial advantage of our open-source MXene HdEEG system compared with commercially available arrays is reduced cost (US$6.64vs US$778).Although a technique has been reported allowing retrieval and reuse of the commercial HdEEG up to six times (Jonak et al., 2018), the cost per use of the commercial array is still substantially greater than that of the MXene HdEEG system.Therefore, our open-source method will be especially beneficial for investigators needing to screen many subjects with chronic EEG.

mTBI acutely increases preictal network connectivity
Although previous studies used epidural EEG2/limited-channel EEG to measure the effects of mTBI in mice (Lim et al., 2013;Modarres et al., 2017;Ichkova et al., 2020;MacMullin et al., 2020), this is the first report, to our knowledge, that measured the effects of mTBI on EEG using non-penetrating electrodes or with HdEEG.We found that although mTBI did not change righting time compared with sham exposure and there were no visible behavior differences between mTBI and sham mice, mTBI did acutely increase SWD preictal network connectivity with the most notable increases in β-spectral frequency.Mild TBI increases the risk of developing epilepsy (Annegers et al., 1998;Christensen et al., 2009;Karlander et al., 2021), and early seizures, those that occur within 7 d of the injury, may increase that risk (Lowenstein, 2009).Because epileptiform SWDs are preceded by increased β-spectral power (Ding et al., 2019;Sorokin et al., 2016, and Fig. 6) increased network connectivity in the β-frequency band (Fig. 7) may raise the risk of early seizures.Future studies will determine the association of mTBI, β-spectral power, and early seizures.

Study limitations
Intermittent recordings demonstrated that although SWD waveform morphology and relative spectral power distributions were the same on Day 21 as on Days 7 and 14, the voltage was reduced, possibly due to increased electrode skull impedance (Fig. 4).Longer studies will need to be performed to determine the maximum duration electrodes provide quality recordings.Moreover, the lifespan experiment (Fig. 4) was performed using intermittent weekly 3 h recordings.Possibly, the MXene electrode lifespan would be reduced even further during continuous recordings with electrode wires causing continuous tension on the surface array.Therefore, future long-duration recordings should be performed continuously and/or with a wireless acquisition system.The mean WPLI/degree prior to sham/mTBI is depicted on the left of the panel and those after sham/mTBI are on the right.Grids depict mean differences in 2-30 Hz network node degree between (C) post and prior sham, (D) post and prior mTBI, and (E) mTBI and sham at all channels at 1.5 s prior to SWD onset.CBP statistically tested differences between sham and mTBI in network node degree changes (p = 0.02) with clusters including most channels at times 1.0 (14-20 Hz) and 1.5 (13-20 Hz) seconds prior to mTBI.The white lines enclose channel/frequency clusters at 1.5 s prior to SWD onset.
This study demonstrated that MXene electrode recordings successfully localized the 6-8 Hz epileptiform discharges to the anterior region (Fig. 6B), captured low-voltage, high-frequency preictal β-activity (Fig. 6C,D), and identified the increased mTBI-associated β-frequency network connectivity (Fig. 7).However, it should be emphasized that epileptiform activity is of higher voltage than physiological rhythms and that the β-frequency activity (Figs. 6, 7) was both generalized and time-locked to SWD onset.Our study did not determine if MXene skull surface electrodes would be sufficient to localize focal low-voltage physiological rhythms as might be obtained in mouse behavioral experiments.In fact, we found that compared with epidural recordings, MXene EEG attenuated theta/alpha frequencies during wakefulness (Fig. 2).Additional studies will need to be performed to test the ability of MXene skull surface EEG to detect low-voltage electrophysiological activity that would otherwise be identified with intracranial electrodes.

Conclusions
In conclusion, this study determined that open-source MXene EEG2 and HdEEG electrodes represent an inexpensive, convenient method for recording mouse EEG sleep/wake cycles, epileptiform activity, and preictal rhythms/connectivity without skull disruption.This represents a refinement in animal care.Using MXene HdEEG recordings, we determined that mTBI increased preictal SWD network connectivity, a change that could increase SWDs early after mTBI.Future studies will determine the long-term longevity of MXene skull surface electrodes in wired and wireless acquisition systems and the capability of these electrodes to detect low-voltage electrophysiological activity typically identified with intracranial electrodes.

Figure 1 .
Figure 1.MXene HdEEG fabrication.A, Drawing of the mouse dorsal cortical surface showing the position of the 16 HdEEG channels (yellow) along with the ground (Grd) and reference (Ref) placed over the midbrain.The uppercase M, S, and V letters of the electrode names indicate placement over motor, somatosensory, and visual cortices.The lowercase a and p letters in the M1/2 and S5/6 electrodes indicate anterior and posterior placement.Electrodes M3 and M4 indicated with red arrows are used for EEG2 recordings.The scale bar indicates 1 mm.B, The circuit diagram shows the HdEEG PCB design.Electrode pads (beige) connected with wires (red = top PCB surface, blue = bottom PCB surface, magenta = separate wires on top and bottom surface) that ultimately terminate on connector pads.Black shapes indicate holes where UV-curable cement is placed.The "B" placed at the bottom of the uppermost black hole indicates positioning over bregma that enables reproducible placement; this is used for visual alignment only.C, Photograph of the bottom of a completed MXene HdEEG array.The silver epoxide is silver, MXene is dark gray, and wires are yellow.D, Intraoperative photograph of implanted MXene HdEEG array.Letter "B" indicates bregma.E, Impedance spectra of 0.1 mm epidural (•) and MXene (▽) electrodes measured in PBS solution (N = 5).

Figure 2 .
Figure 2. Epidural and MXene EEG2 recordings of wakefulness and SWS.A,B, Sample 24 h EEG spectral power, δ/ϴ power ratio, and EMG spectral density plots from EEG2 recordings obtained with Pinnacle amplifiers 7 d after implantation with 1.0 mm epidural (A) and MXene (B) electrodes.The dashed blue lines indicate 4 Hz.Time periods with increased EMG power, decreased delta (≤4 Hz) spectral power, and decreased δ/ϴ ratio indicate wakefulness (examples marked with a single dagger, †).Periods with decreased EMG power, increased delta (≤4 Hz) spectral power, and increased δ/ϴ ratio indicate SWS (examples marked with a double dagger, ‡).EMG associated with the Pinnacle epidural electrodes and those with the MXene electrodes are on different power scales because Pinnacle EMG wires are uninsulated while the MXene EMG wires are insulated except at the tips.Sample EEG2 recordings obtained 7 d after implantation of wakefulness (C,E) and SWS (D,F) with (C,D) 1.0 mm epidural electrodes (Pinnacle left frontal electrode referenced to the cerebellum) and (E,F) MXene electrodes (channel M3 −1.85 mm M/L, −0.25 mm A/P referenced to the midbrain, 11 kΩ skull impedance).The δ/ϴ ratio values for awake and SWS samples are given in each panel.Mean awake (G,H) SWS spectral power ± SEM obtained with epidural (•, N = 6) and MXene (▽, N = 5) electrodes.Insets in G and H depict the spectral power of Z-transformed EEG.Fs = 200 Hz, BP 0.5-55 Hz.

Figure 5 .
Figure 5. Epidural and MXene EEG2 recordings of tonic-clonic seizures Sample PTZ-evoked tonic-clonic seizures recorded with Pinnacle amplifiers 7 d after implantation with (A) 1.0 mm epidural (Pinnacle left frontal electrode referenced to the cerebellum) or (B) MXene (channel M3 −1.85 mm M/L, −0.25 mm A/P referenced to the midbrain, 10 kΩ skull impedance) electrodes.The bottom traces in A and B depict the first 5 s of the seizures on an expanded timescale.C, Mean ± SE spectral power of the first 5 s of tonic-clonic seizures obtained with epidural (•, N = 5 mice) and MXene (▽, N = 5 mice) electrodes and the inset is the spectral power of the Z-transformed EEG.Fs 200 Hz, BP 0.5-55 Hz.

Figure 7 .
Figure7.Effects of mTBI on preictal SWD network connectivity.A,B, Mean 13 Hz network node degree (colored circles at electrode positions) and WPLI connectivity (red lines between the electrodes, line intensity corresponds to WPLI values) at 1.5 s before SWD for mice that underwent (A) sham exposure (N = 7 mice) or (B) mTBI (N = 8 mice).The mean WPLI/degree prior to sham/mTBI is depicted on the left of the panel and those after sham/mTBI are on the right.Grids depict mean differences in 2-30 Hz network node degree between (C) post and prior sham, (D) post and prior mTBI, and (E) mTBI and sham at all channels at 1.5 s prior to SWD onset.CBP statistically tested differences between sham and mTBI in network node degree changes (p = 0.02) with clusters including most channels at times 1.0 (14-20 Hz) and 1.5 (13-20 Hz) seconds prior to mTBI.The white lines enclose channel/frequency clusters at 1.5 s prior to SWD onset.